PCR Test for the Detection of Bluetongue All + Type 8

1. Purpose of the test
ADIAVET™ BTV All + TYPE 8 REAL TIME kit is intended to detect, in once run, the 27 serotypes of
Bluetongue Viruses (BTV) and to identify the serotype 8 specifically, using real-time Polymerase Chain
Reaction (PCR) technology, from whole blood specimen of bovine and ovine.
2. Pathogen
The bluetongue virus is a non-contagious viral arthropod-borne infectious disease due to an Orbivirus
(family Reoviridae, virus ARN), mainly transmitted by hematophageous midges from Culicoides genus.
The disease is found in countries where these midges are prevalent and clinical cases have been
reported in Africa, the Middle East, the USA, Asia and southern Europe. It induces serious syndromes by
ovine (fever, oedema, slimming, mortality 1 to 10%), but it is mainly asymptomatic by caprine, domestic
or wild ruminants, which are the virus reserve.
Once thought to be restricted in Africa and parts of the Middle East, bluetongue has now become a
concern in sheep-rearing countries around the world. In the past 10 years, severe outbreaks have
occurred in Europe with important economic consequences. The 2006-2008 outbreak in Europe was
caused by a serotype 8 strain and the 2014 outbreak in Greece and the other countries of south-east
Europe was caused by a serotype 4 strain, suggested a high activity of the Culicoïdes vector.
The clinical expression is widely dependent on the environmental parameters (nutritional state,
parasitism and bacterial infections concomitant) and on the individual sensitivity. 26 distinct serotypes
exist inducing partial or no cross protections between them.
Under the natural conditions, the dissemination is exclusively the fact of infected biting midge or the
seed of infected males. The diffusion of the disease thus is largely influenced by the activity of the
midge.
Transmission by pregnant ewes has also been described. Transmission by contaminated blood injection
is possible when needles and syringes are re-used.
Samples for virus detection are bloods of animals with anticoagulants (EDTA). Virus is detected by
isolation on embryonated eggs, in vitro cell culture, immunofluorescence on cell culture or by PCR.
3. Description and purpose of the test
This test is based first on the reverse transcription (RT) of RNA into complementary DNA. Then, cDNA is
amplified (PCR) by a DNA polymerase using specific primers. Both enzymatic reactions occur in the
same tube (One-step RT-PCR).
Amplified products are detected in real-time thanks to a specific labelled hydrolysis probe (5’-
exonulease technology).
The ADIAVET™ BTV All + TYPE 8 REAL TIME kit enables the simultaneous detection of:
– The Bluetongue Virus (probe labelled in FAM),
– The Bluetongue Virus Type 8 (probe labelled in VIC),
– The GAPDH, an internal control of extraction and amplification steps specific from an
endogenous RNA (probe labelled in CY5).
ADIAGENE validated the test using RNA purification kits (Qiagen, Macherey-Nagel). Other purification
kits can be used if they have been validated by the user.